The effect of caffeic acid phenethyl ester (CAPE) on metabolic enzymes including acetylcholinesterase,butyrylcholinesterase, glutathione S-transferase,lactoperoxidase, and carbonic anhydrase isoenzymes I,II, IX,and XII

Caffeic acid phenethyl ester (CAPE) is an active component of honeybee propolis extracts. Carbonic anhydrases (CAs, EC 4.2.1.1) are widespread and intensively studied metalloenzymes present in higher vertebrates including humans as many diverse isoforms. Acetylcholinesterase (AChE) is responsible for acetyl choline (ACh) hydrolysis and plays a fundamental role in nerve impulse transmission by terminating the action of the ACh neurotransmitter at cholinergic synapses and neuromuscular junctions. Butyrylcholinesterase (BChE) is another enzyme abundantly present in the liver and released into blood in a soluble form. Lactoperoxidase (LPO) is an enzyme involved in fighting pathogenic microorganisms whereas glutathione S-transferases (GSTs) are dimeric proteins present both in prokaryotic and eukaryotic organisms and involved in cellular detoxification mechanisms. In the present study, the inhibition effect of CAPE on human carbonic anhydrase (hCA) isoforms I, II, IX, and XII, AChE, BChE, LPO, and GST was evaluated. CAPE inhibited these enzymes with K_is in the range between micromolar to picomolar. The best inhibitory effect was observed against AChE and BChE.     

Functional mimetic of the G-protein coupled receptor CXCR4 on a soluble antibody scaffold

G-protein coupled receptors (GPCRs) constitute major drug targets due to their involvement in critical biological functions and pathophysiological disorders. The leading challenge in their structural and functional characterization has been the need for a lipid environment to accommodate their hydrophobic cores. Here, we report an antibody scaffold mimetic (ASM) platform where we have recapitulated the extracellular functional domains of the GPCR, C-X-C chemokine receptor 4 (CXCR4) on a soluble antibody framework. The engineered ASM molecule can accommodate the N-terminal loop and all three extracellular loops of CXCR4. These extracellular features are important players in ligand recruitment and interaction for allostery and signal transduction. Our study shows that ASM^CXCR4 can be recognized by the anti-CXCR4 antibodies, MEDI3185, 2B11, and 12G5, and that ASM^CXCR4 can bind the HIV-1 glycoprotein ligand gp120, and the natural chemokine ligand SDF-1α. Further, we show that ASM^CXCR4 can competitively inhibit the SDF-1α signaling pathway, and be used as an immunogen to generate CXCR4-specific antibodies. This platform will be useful in the study of GPCR biology in a soluble receptor context for evaluating its extracellular ligand interactions.     

Fine mapping of the Schnyder’s crystalline corneal dystrophy locus

Schnyders crystalline corneal dystrophy (SCCD) is a rare autosomal dominant eye disease with a spectrum of clinical manifestations that may include bilateral corneal clouding, arcus lipoides, and anterior corneal crystalline cholesterol deposition. We have previously performed a genome-wide linkage analysis on two large Swede-Finn families and mapped the SCCD locus to a 16-cM interval between markers D1S2633 and D1S228 on chromosome 1p36. We have collected 11 additional families from Finland, Germany, Turkey, and USA to narrow the critical region for SCCD. Here, we have used haplotype analysis with densely spaced microsatellite markers in a total of 13 families to refine the candidate interval. A common disease haplotype was observed among the four Swede-Finn families indicating the presence of a founder effect. Recombination results from all 13 families refined the SCCD locus to 2.32 Mbp between markers D1S1160 and D1S1635. Within this interval, identity-by-state was present in all 13 families for two markers D1S244 and D1S3153, further refining the candidate region to 1.58 Mbp.     

A combined allele-specific PCR and RFLP assay to detect the 35delG mutation in the Connexin 26 gene

Mutations in the Connexin-26 (specified GJB2) gene have been shown to be a major cause of nonsyndromic recessive deafness (NSRD), and a single mutation 35delG in the GJB2 gene accounts for the majority of cases of NSRD. For diagnostic analyses and for scientific studies of large numbers of patients, fast and economic assays that can be performed with standard polymerase chain reaction (PCR) instruments are highly desirable. We have developed an allele-specific amplification (ASA)-based restriction fragment length polymorphism (RFLP) assay. We evaluated the multiplex method for its ability to 35delG mutation. Our method is a stable, reproducible and concordend with previously reported PCR-RFLP assays.     

Acute effect of hemodialysis on serum levels of the proinflammatory cytokines

Chronic inflammation is a common feature of end-stage renal disease, which carries a heightened risk of atherosclerosis and other co-morbid conditions. Dialysis treatment per se can bring additional risk factors for inflammation, such as increased risk of local graft and fistula infections, impure dialysate or bio-incompatible membranes. Our study was designed to determine whether a hemodialysis session leads to an acute substantial alteration in the plasma levels of the proinflammatory cytokines interleukin (IL)-6, IL-1beta and tumor necrosis factor (TNF)-alpha, the T-lymphocyte activation factor soluble IL-2 receptor (sIL-2R), and an inflammation mediator and chemotactic granulocyte factor, IL-8, in end-stage renal disease patients receiving chronic intermittent HD. In this study, 21 (12 male/nine female) patients undergoing chronic hemodialysis were enrolled. The acute effect of a hemodialysis session on serum cytokine concentrations was assessed by comparison of pre-hemodialysis and post-hemodialysis determinations. Serum IL-1beta, sIL-2R, IL-6, IL-8 and TNF-alpha levels were determined with chemiluminescence enzyme immunometric assays. A significant difference was not observed for IL-1beta, IL-6, TNF-alpha, and sIL-2R concentrations in pre-hemodialysis and post-hemodialysis specimens (p>0.05). Serum median (25th-75th percentiles) IL-8 concentration was 69.4 (34.9-110.3) pg/ml before hemodialysis, and decreased to 31.5 (18.0-78.8) pg/ml following hemodialysis (p: 0.006). Clearance of IL-8 increased by 0.47+/-0.08 pg/ml for each unit increase in pre-dialysis IL-8 (p<0.001) and decreased by 5.63+/-2.59 pg/ml for each unit increase in pre-dialysis urea mmol/l (p<0.05). In conclusion, the results of our study demonstrate that a hemodialysis session markedly decreases IL-8 concentration, which is significantly affected by pre-dialysis concentrations, indicating that removal of IL-8 is a concentration gradient-dependent action, but does not change the serum levels of IL-1beta, sIL-2R, IL-6, and TNF-alpha, underlining importance of the structural characteristics of the molecules.     

Mutational analysis of ATP-grasp residues in the two ATP sites of Saccharomyces cerevisiae carbamoyl phosphate synthetase

The ATP-grasp fold is found in enzymes that catalyze the formation of an amide bond and occurs twice in carbamoyl phosphate synthetase. We have used site-directed mutagenesis to further define the relationship of these ATP folds to the ATP-grasp family and to probe for distinctions between the two ATP sites. Mutations at D265 and D810 severely diminished activity, consistent with consensus ATP-grasp roles of facilitating the transfer of the gamma-phosphate group of ATP. H262N was inactive whereas H807N, the corresponding mutation in the second ATP domain, exhibited robust activity, suggesting that these residues were not involved in the ATP-grasp function common to both domains. Mutations at I316 were somewhat catalytically impaired and were structurally unstable, consistent with a consensus role of interaction with the adenine and/or ribose moiety of ATP. L229G was too unstable to be purified and characterized. S228A showed essentially wild-type behavior.     

A case of Lemierre's syndrome following Epstein-Barr virus infection

Lemierre's syndrome, or necrobacillosis, is a life-threatening septic thrombophlebitis of the internal jugular vein. The causative organism is Fusobacterium necrophorum. Here we report a case of Lemierre's syndrome in a young male and describe the clinical presentations and treatment. Epstein-Barr virus (EBV) was a suspected predisposing factor in this case.     

Tall cell variant of papillary carcinoma arising from a thyroglossal cyst: report of a case with diagnosis by fine needle aspiration cytology

BACKGROUND: Papillary carcinoma arising from a thyroglossal duct cyst (TDC) is the most frequent malignancy arising from this embryologic remnant of the thyroglossal duct. The preoperative fine needle aspiration cytology (FNAC) of such neoplasms has rarely been cited. Approximately 170 TDC cases have been reported in the literature worldwide, diagnosed solely on histopathology. CASE: A 63-year-old woman presented with an anterior midline neck mass of years' duration. A hypodense cyst was located at the level of the hyoid bone. FNAC of the midline neck mass showed bloody, mucoid material and numerous atypical, elongated cells. The aspirated material was reported as "papillary carcinoma possibly arising from the thyroglossal duct," and the diagnosis was confirmed by histopathologic investigation on resection material. CONCLUSION: Tall cell variant is a rare variant of papillary carcinoma and has a poor prognosis. The differential diagnosis includes Hürthle cell lesions, oncocytic papillary thyroid carcinoma and Warthin-like papillary carcinoma.